Approximately 20% of human proteins are predicted to contain disulfide bonds between cysteine residues. [1] Small-molecule thiols can reduce these (and other) disulfide bonds, thereby modulating biomolecular function. [2] The reaction mechanism involves thiol-disulfide interchange initiated by a thiolate. [3] The ensuing mixed disulfide, however, can become trapped if the reagent is a monothiol, such as β-mercaptoethanol (βME). [4]
To overcome this problem, Cleland developed racemic (2S,3S)-1,4-dimercaptobutane-2,3-diol (dithiothreitol or DTT; Table 1), a dithiol that resolves a mixed disulfide by forming a six-membered ring. [2a, 5] DTT is a potent reducing agent (E°′—0.327 V) [2g] and has been, despite its high cost, the preferred reagent for the quantitative reduction of disulfide bonds and is now the standard reagent for reducing disulfide bonds in biological molecules. [6, 7] At physiological pH, DTT is, however, a sluggish reducing agent. The reactivity of a dithiol is governed by the lower of its two thiol pKa values. [2, 3] With its lower thiol pKa value being 9.2, [Table 1] greater than 99% of DTT thiol groups are protonated at pH 7 and thus unreactive (i.e., less than 1% of DTT residues are in the reactive thiolate form at pH 7 [8])
Thus, there is a need in the art for reducing agents useful in biological systems, for example, for the reduction of disulfide bonds, which exhibit properties improved over those of prior art reducing agents. The present invention provides dithiol amines which can be prepared from inexpensive starting materials in high yield and which exhibit desirable improved properties as reducing agents.